title: Proteomics-based identification of novel factor inhibiting hypoxia-inducible factor (FIH) substrates indicates widespread asparaginyl hydroxylation of ankyrin repeat domain-containing proteins.
abstract: Post-translational hydroxylation has been considered an unusual modification on intracellular proteins. However, following the recognition that oxygen-sensitive prolyl and asparaginyl hydroxylation are central to the regulation of the transcription factor hypoxia-inducible factor (HIF), interest has centered on the possibility that these enzymes may have other substrates in the proteome. In support of this certain ankyrin repeat domain (ARD)-containing proteins, including members of the IkappaB and Notch families, have been identified as alternative substrates of the HIF asparaginyl hydroxylase factor inhibiting HIF (FIH). Although these findings imply a potentially broad range of substrates for FIH, the precise extent of this range has been difficult to determine because of the difficulty of capturing transient enzyme-substrate interactions. Here we describe the use of pharmacological "substrate trapping" together with stable isotope labeling by amino acids in cell culture (SILAC) technology to stabilize and identify potential FIH-substrate interactions by mass spectrometry. To pursue these potential FIH substrates we used conventional data-directed tandem MS together with alternating low/high collision energy tandem MS to assign and quantitate hydroxylation at target asparaginyl residues. Overall the work has defined 13 new FIH-dependent hydroxylation sites with a degenerate consensus corresponding to that of the ankyrin repeat and a range of ARD-containing proteins as actual and potential substrates for FIH. Several ARD-containing proteins were multiply hydroxylated, and detailed studies of one, Tankyrase-2, revealed eight sites that were differentially sensitive to FIH-catalyzed hydroxylation. These findings indicate that asparaginyl hydroxylation is likely to be widespread among the approximately 300 ARD-containing species in the human proteome.
authors.affiliation: Central Proteomics Facility, University of Oxford, Oxford, United Kingdom.
authors.forename: Matthew E
authors.initials: ME
authors.lastname: Cockman
authors.forename: James D
authors.initials: JD
authors.lastname: Webb
authors.forename: Holger B
authors.initials: HB
authors.lastname: Kramer
authors.forename: Benedikt M
authors.initials: BM
authors.lastname: Kessler
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Ratcliffe
mesh_terms: D000595:Amino Acid Sequence; D000600:Amino Acids, Dicarboxylic / Q000494:pharmacology; D017089:Ankyrin Repeat; D001216:Asparagine / Q000378:metabolism*; D045744:Cell Line, Tumor; D004722:Endoribonucleases / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006900:Hydroxylation / Q000187:drug effects; D015151:Immunoblotting; D013058:Mass Spectrometry; D006899:Mixed Function Oxygenases; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000187:drug effects; D040901:Proteomics / Q000379:methods*; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D015203:Reproducibility of Results; D013379:Substrate Specificity / Q000187:drug effects; D038501:Tankyrases / Q000737:chemistry / Q000378:metabolism
chemical_list: D000600:Amino Acids, Dicarboxylic; D012097:Repressor Proteins; D001216:Asparagine; D006899:Mixed Function Oxygenases; C439790:HIF1AN protein, human; D038501:Tankyrases; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease; C040947:oxalylglycine